Enzyme assays, such as enzyme immunoassays (EIA), use enzymes as markers to quantitatively follow antigen-antibody reactions. These assays have been described in a multitude of publications like Enzyme Immunoassay, Ed., Ishikawa et al., Igaku-Shoin Ltd., Tokyo (1981).
Reagents required such as enzyme-labeled antigens and antibodies or enzyme-labeled nucleic acids are usually stable in a conventional refrigerator for years. No expensive equipment is required. These assays can be performed without much experience in the laboratory and pose no problem with respect to disposing hazardous radioactive waste materials. Indeed, enzyme assays are convenient and powerful analytical tools in a variety of research fields and clinical diagnostics.
When an enzyme is used as a label, it should be easily recognizable, stable and have catalytic activity which produces a detectable change in the substrate for that enzyme. In other words, the enzyme should maintain sufficient activity and, at the same time, should function satisfactorily under the conditions in which an assay is performed. In addition, the label must not disintegrate or deteriorate.
To be used as a label, the number of enzyme molecules must be measured quantitatively. An enzyme labeled binding assay method is based on counting the number of enzymes which are linked to antigen, antibody, or nucleic acid molecules instead of counting the latter molecules directly. However, the number of label enzyme molecules themselves cannot be counted, rather, the enzyme activity of the label is counted and the number of labels that have catalyzed the reaction can be determined. Therefore, an enzyme assay depends upon the assumption that the catalytic activity that is obtained from the physical measurement is proportional to the amount of enzyme that catalyzes this reaction.
Such measurements of enzyme activity can be affected by the pH of a solution. Thus, virtually all quantitative assays performed in aqueous solution are carried out in the presence of a buffer to control pH. A buffer is a substance which, when added to a solution, resists a change in hydrogen ion concentration on addition of acid or alkali. Selection of an appropriate buffer depends on a variety of factors such as the pH range over which it is effective, solubility, purity, stability, etc.
Since quantitative measurements often involve optical determinations by measuring changes in absorbance or emission, useful buffers should not interfere substantially with the optical determination in the Wavelength region of measurement.
Alkanol amine buffers such as triethanolamine (TEA) or diethanolamine (DEA) are used quite commonly in clinical analyses because they have useful buffer ranges, are substantially optically transparent, and generally do not prove harmful to physiological substances like enzymes, antigens, antibodies or nucleic acids.
However, such buffers have a limited shelf life. Over time, these alkanol amine buffers can form degradation products which substantially interfere with optical determinations in the wavelength region of measurement.